Littlefield Kimberly Palmiter, Swank Douglas M, Sanchez Becky M, Knowles Aileen F, Warshaw David M, Bernstein Sanford I
Department of Biology, Molecular Biology Institute, San Diego State University, San Diego, California 92182-4614, USA.
Am J Physiol Cell Physiol. 2003 Apr;284(4):C1031-8. doi: 10.1152/ajpcell.00474.2002. Epub 2002 Dec 11.
Recently the converter domain, an integral part of the "mechanical element" common to all molecular motors, was proposed to modulate the kinetic properties of Drosophila chimeric myosin isoforms. Here we investigated the molecular basis of actin filament velocity (V(actin)) changes previously observed with the chimeric EMB-IC and IFI-EC myosin proteins [the embryonic body wall muscle (EMB) and indirect flight muscle isoforms (IFI) with genetic substitution of the IFI and EMB converter domains, respectively]. In the laser trap assay the IFI and IFI-EC myosins generate the same unitary step displacement (IFI = 7.3 +/- 1.0 nm, IFI-EC = 5.8 +/- 0.9 nm; means +/- SE). Thus converter-mediated differences in the kinetics of strong actin-myosin binding, rather than the mechanical capabilities of the protein, must account for the observed V(actin) values. Basal and actin-activated ATPase assays and skinned fiber mechanical experiments definitively support a role for the converter domain in modulating the kinetic properties of the myosin protein. We propose that the converter domain kinetically couples the P(i) and ADP release steps that occur during the cross-bridge cycle.
最近，转换结构域作为所有分子马达共有的“机械元件”的一个组成部分，被认为可调节果蝇嵌合肌球蛋白同工型的动力学特性。在此，我们研究了先前在嵌合EMB-IC和IFI-EC肌球蛋白蛋白（分别是胚胎体壁肌肉（EMB）和间接飞行肌肉同工型（IFI），其IFI和EMB转换结构域进行了基因替换）中观察到的肌动蛋白丝速度（V(actin)）变化的分子基础。在激光阱测定中，IFI和IFI-EC肌球蛋白产生相同的单位步移（IFI = 7.3 +/- 1.0纳米，IFI-EC = 5.8 +/- 0.9纳米；平均值 +/- 标准误）。因此，转换结构域介导的强肌动蛋白-肌球蛋白结合动力学差异，而非蛋白质的机械能力，必定是观察到的V(actin)值的原因。基础和肌动蛋白激活的ATP酶测定以及皮纤维力学实验明确支持转换结构域在调节肌球蛋白蛋白动力学特性中的作用。我们提出，转换结构域在动力学上耦合了横桥循环中发生的无机磷酸（P(i)）和二磷酸腺苷（ADP）释放步骤。
